Enzymatic Disaggregation, Collagenase, Maligant Tissue, intracellular Matrix, embryonic Normal, Primary Cell Culture, Epithelial Cells

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	Enzymatic Disaggregation The two important enzymes used in tissue disaggregation are collagenase and trypsin. 1. CollagenaseIt is used for disaggregation of embryonic, normal and malignant tissues, by digesting the collagen of intracellular matrix Tisue Disaggregation By Collagenase
Dissection in BSS+Antibiotic Wash by setling Collagenase in complete medium(1-5 days)
Pipetting to disperse tissue Separation of residual clusters of epithelial cells by settling Clusters Washed Settling Enriched Fiberblast fraction plated out Enriched epithelial fraction plated out Tissue Disaggregation by Collagenase.
The biopsy tissues are kept in medium' containing antibiotics. It is then dissected into pieces in basal salt solution containing antibiotics. The chopped tissue is properly washed with sterile distilled water and transferred into complete medium containing collagenase. After 5 days of treatment, the mixture is pipetted so that the medium may get dispersed. When the whole treatment is left for sometime, the cluster of cells settles at the bottom of the test tubes. The cluster present in the test tubes is washed either by settling or is centrifuged to make the dispersed cell suspension free from collagenase. The cell suspension now consists of enriched fibroblast fraction which is plated out on medium. The cluster which is washed by settling, contains enriched epithelial fraction. I t is also plated out on medium.
	2.TrypsinUse of trypsin for disaggregation of tissue is called trypsinization. It may be of two types: 1. Warm trypsinization and 2. Cold trypsinization. Crude trypsin is the most common enzyme used for disaggregation. It is tolerated by a variety of cells and is effective for many tissues. Its residual activity is neutralized by the serum of the medium or by a trypsin inhibitor (e.g. Soybean trypsin inhibitor), in the case of serum-free medium. The cells are exposed to the warm enzyme (36.5°C) for a minimum period. The dissociated cells are collected every half an hour. The trypsin is removed by centrifugation after 3-4 hours, which is required for complete disaggregation. Cold trypsinization involves soaking of tissue in trypsin at O°C to allow penetration of enzyme, followed by incubation at 36.5°C for a shorter period.